Microalgae have been promoted as one of the most ideal candidates for biofuel production. Although energy can be obtained from the microalgal biomass directly by pyrolysis or other methods, the production of liquid fuels is by far the most sought after form of energy. Lipids are the most energy-dense compounds for liquid fuel production. However, lipids must be extracted from the microalgae before the lipids may be used for fuel.
The most common methods for lipid extraction from microalgal biomass use solvents and are adapted from the procedures used for other sources, including oil seeds. Lipid extraction often requires large amounts of solvent, high temperatures, pressure and/or agitation due to cell walls of microalgae which significantly limit contact between the lipid and the solvent. Solvent extraction is limited by the extent to which the cell wall of the microorganism can be penetrated. If solvents cannot reach the lipids inside the cell, they cannot extract them. There are various methods to disrupt or rupture cell walls, including molecular methods, enzymes, detergents, and mechanical methods, which require several cleaning steps to remove the chemicals used. Heat may be applied to microalgae for some extractions, but some target chemical compounds may not be not heat stable. High temperatures used over long extraction times may lead to unwanted oxidation of fatty acids.
There are two main strategies used for solvent extraction: (1) reflux of heated solvents through the biomass, and (2) the immersion of the biomass in solvents in various proportions. The Soxhlet method, which uses solvent reflux, can be efficient for dry biomass, but requires extensive preparation of the biomass for extraction. Researchers have found that enzymatic degradation of lipids (lipolysis, oxidation) can occur before and during extraction of lipids from microalgal and cyanobacterial biomass, particularly if water is present. The Bligh-Dyer and the Folch methods are used mainly for wet biomass and are based on solvent immersion.
While these are established methods, there are numerous issues with their implementation for microalgal lipid extraction. For example, the selection of the solvent for these methods can impact the final recovery of the lipids. Further, large volumes of solvent are needed for an efficient extraction of the lipids creating a potential environmental issue.
Chloroform and hexane are commonly used for solvent extraction. With these solvents, a sequence of extractions may be needed to obtain a large percentage of the lipids. As an example, it has been determined that a two-step extraction with acetone and a five-step separation with a water-alcohol-hexane system was needed to extract 80-90% of the non-polar lipids from the biomass of a species of Scenedesmus. Extraction efficiencies below 50% have been reported in algal species with thick cell walls.
Solvents are used not only for lipid extraction, but also for pigment extraction. The most common solvents for this application are methanol, ethanol, dimethylformamide (DMF), acetone, and hexane. The solvent extraction of photosynthetic pigments, as in the case of lipids, requires an efficient contact of the solvents with a pigment source. Also, pigment-degrading enzymes such as chlorophyllases, which maintain activity after harvesting, can rapidly decrease the chlorophyll content of a biomass in the time between harvesting and extraction.
Extraction methods may also be employed to extract proteins from organisms with resistant cell walls. Current methods include exposure grinding with glass beads, mechanical homogenization, sonication, and others, which yield mixed results. Incomplete extraction may lead to inexact estimation of protein content in a biomass. Additionally, some procedures require long treatment times that can lead to protein degradation.
An important aspect of various chemical compound extraction methods from microalgal species having thick cell walls is rupture of the cell wall to facilitate release of the chemical compounds. Because of the issue of cell wall thickness, numerous studies have been performed to investigate pre-treatment options as a means to enhance the extraction process. These include microwave, freezing, sonication, bead beating, autoclaving, electroporation, ultrasound, high pressure homogenization and cycling, and chemical cell lysis.
The effectiveness of a cell disruption method will vary depending on the characteristics of the cells to be treated. For example, cells with a high cellulose content, and hence with more robust cell walls (i.e., Chlorella, and Scenedesmus) will be more difficult to rupture than more fragile cells like Chlamidomonas, Spirulina, Scytonema or Rhyzoclonium, which have low or no sporopollenin in their cell wall. It has been discovered that microwave treatment increases the lipid yield two- to threefold when compared with sonication for Botryococcus braunii, but autoclaving resulted in the highest efficiency for lipid extraction from Chlorella vulgaris, which has a more resistant cell wall.
Methods that require large inputs of energy or long pretreatment times present challenges due to potential degradation of the target products and negative impacts on cost and energy balance. Enzymatic reactions have also been used for cell wall destruction of immobilized cells. However, the immobilization of the cells, the cost of the enzymes, and the possible effects on the lipids and pigments may be problematic in large scale use of this method.
Chemical lysis is an efficient method for cell wall rupture, but may not be suitable for sensitive products, as harsh chemicals such as strong acids or alkalis can destroy target compounds. In-situ extraction from live cells has been investigated as an alternative for cell harvesting and extraction, but it was found that the product recovery was due to the lysis of the cells and not separation of pigments and lipids from live cells, as originally hypothesized.
Other methods, such as thermochemical liquefaction, microwave-assisted extraction, and supercritical fluid extraction have gained some interest as alternatives to direct solvent extraction. The thermochemical liquefaction has the advantage of extracting lipids from wet biomass, reducing processing time. However, the process requires high temperatures and pressures. Microwave-aided extraction presents some advantages, as it allows a shorter extraction time and has shown good efficiency, particularly in multistep processes. Extraction of lipids with supercritical fluids, including carbon dioxide, has been performed with biomasses of different crops, with mixed results. Although this method has been tested for lipid and pigment extraction, the use of CO2 as the carrier for oil extraction requires high pressures (and high reactor cost), representing a challenge for wide scale application.
The currently-used technologies for pretreatment and chemical compound extraction from microalgae and other organisms, by the various methods described, including solvent extraction, milking, and pretreatment of biomass, lack efficiency (energetic, economic and/or product quality), thus increasing the final cost of the pigments and oil.
Therefore, there is a need for extraction methods that increase efficiency and reduce environmental concerns, thereby reducing costs associated with extraction, including extraction of biofuels, pigments, proteins and other bio-products.